L-[1-^(13)C]苯丙氨酸呼气试验定量检测肝脏功能动物实验研究

Experimental study on L-[1-^(13)C] phenylalanie breath test for quantitative assessment of liver function with animal

目的采用自行设计的小动物呼气试验模型进行大鼠L-[1-13C]苯丙氨酸呼气试验(13C- PheBT),以验证该实验方法的可行性和有效性,并提供有效的试验参数。方法280～290g的雄性SD 大鼠20只,随机分为急性肝损伤组和正常对照组,每组10只,采用四氯化碳橄榄油灌胃染毒复制急性肝损伤模型,小动物呼气机进行机械通气制作呼气试验模型;按20mg／kg体重尾静脉弹丸给予13C- 苯丙氨酸(13C-Phe),收集给药前和给药后1～60min呼出气样共29次气样,应用气体同位素比值质谱仪测定样品中13C丰度。结果13C排除时相曲线呈单峰,峰值多位于给药后2min;急性肝损伤大鼠呼气试验参数13C排除速率常数(PheBT-K)为(2．45±0．25)×10-2min-1,显著低于正常对照组(2．98±0．19) ×10-2min-1(t=5．40,P<0．001),而急性肝损伤大鼠呼出气中13C排除峰值和血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST、总胆汁酸(TBA)、碱性磷酸酶(AKP)和总胆红素(TBIL)含量均显著高于正常对照组(t值分别为8．15,3．40,3．90,4．83和4．12,P<0．05);13C快处置常数在两组间差异无显著性(t= 0．58,P>O．05);急性肝损伤大鼠PheBT-K与血清ALT和AKP活度以及TBA和TBIL水平呈负相关(r 分别为-0．74、-0.73、-0．82和-0．67,P值均小于O．05),而与血清AST活度无相关性(r=0．16,p> 0．05)。结论自行设计的小动物呼气试验模型是进行呼气试验基础研究的有效工具;动物PheBT-K 是一项灵敏的分析指标。

Objective Using a small animal breath test model we designed and L-[1-^13C] phenylalanine breath test （^13C-PheBT） of rats, we investigated its feasibility and validity and determined effective parameter of the test. Methods Twenty male Sprague-Dawley（SD） weighting 280～290g rats randomized into two groups acute hepatitis rats （n=10） and control rats （n=10）. Hepatitis was induced by carbon tetrachloride （CCl4）olive oil administration through intragastfic gavage. PheBT was assisted by small mechanical ventilator improved and air samples were collected discontinuously, 20mg/kg body weight L-[1-^13C] phenylalanine （^13C-Phe）was administered intravenously. Twenty-nine breath samples were taken before and different intervals within sixty minutes after administration, ^13C enrichment was measured by isotope ratio mass spectrometer. Results All time phase curves of ^13C enrichment in rat breath reached a peak almost at 2 rain after the intravenous administration of ^13C-Phe. The PheBT parameters, ^13C excretion rate constant （PheBT-K）, of CCl4 hepatitis rats were significantly lower than that of normal control rats[ （2.45±0.25） × 10^-2 min^-1 vs （2.98±0.19）×10^-2min^-1, t=5.40, P〈0.001 ]. Serum alanine transaminase （ALT）, aspartate transaminase （AST）, total bile acid （TBA）, alkaline phosphatase（AKP） in hepatitis rats were significant higher than that of controls （ t value is 8.15, 3.40, 3.90, 4.8 and 4.12 respectively, P〈0.05）. However ^13C fast phase disposition constant did not statistically differ between the two groups （ t=0.58,P〉0.05 ）. PheBT-K had significant negative cor-relation with serum ALT, AKP, TBA and total bilirum TBIL （ the correlation coefficient r is-0.74, -0.73, -0.82 and -0.67 respectively, P〈0.05）, and no statistically significant correlation with serum level of AST（r=0.16, P 〉 0.05）. Conclusions It was indicated that the small animal breath test model we designed was a virtual tool to use in experimental study on breath test and PheBT-K was a sensitive index.